This invention relates to active derivatives of polyethylene glycol and related hydrophilic polymers and to methods for their synthesis for use in modifying the characteristics of surfaces and molecules. The invention also relates to polypeptides that have been covalently bonded to such active derivatives and methods for making the same.
Polyethylene glycol (xe2x80x9cPEGxe2x80x9d) has been studied for use in pharmaceuticals, on artificial implants, and other applications where biocompatibility is of importance. Various derivatives of PEG have been proposed that have an active moiety for permitting PEG to be attached to pharmaceuticals and implants and to molecules and surfaces generally. For example, PEG derivatives have been proposed for coupling PEG to surfaces to control wetting, static buildup, and attachment of other types of molecules to the surface, including proteins or protein residues.
PEG derivatives have also been proposed for affinity partitioning, for example, of enzymes from a cellular mass. In affinity partitioning, the PEG derivative includes a functional group for reversible coupling to an enzyme that is contained within a cellular mass. The PEG and enzyme conjugate is separated from the cellular mass and then the enzyme is separated from the PEG derivative, if desired.
In still further examples, coupling of PEG derivatives (xe2x80x9cPEGylationxe2x80x9d) is desirable to overcome obstacles encountered in the clinical use of biologically active molecules. Published PCT Publication No. WO 92/16221 states, for example, that many potentially therapeutic proteins have been found to have a short half life in the blood serum. For the most part, proteins are cleared from the serum through the kidneys. The systematic introduction of relatively large quantities of proteins, particularly those foreign to the human system, can give rise to immunogenic reactions that, among other problems, may lead to rapid removal of the protein from the body through formation of immune complexes. For other proteins, solubility and aggregation problems have also hindered the optimal formulation of the protein.
PEGylation decreases the rate of clearance from the bloodstream by increasing the apparent molecular weight of the molecule. Up to a certain size, the rate of glomerular filtration of proteins is inversely proportional to the size of the protein. The ability of PEGylation to decrease clearance, therefore, is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Decreased clearance can lead to increased efficiency over the non-PEGylated material. See, for example, Conforti et al., Pharm. Research Commun. vol. 19, pg. 287 (1987) and Katre et al., Proc. Natl. Acad. Sci. U.S.A. vol. 84, pg. 1487 (1987).
In addition, PEGylation can decrease protein aggregation (Suzuki et al., Biochem. Biophys. Acta vol. 788, pg. 248 (1984)), alter protein immunogenicity (Abuchowski et al., J. Biol. Chem. vol. 252 pg. 3582 (1977)), and increase protein solubility as described, for example, in PCT Publication No. WO 92/16221.
PEGylation of proteins illustrates some of the problems that have been encountered in attaching PEG to surfaces and molecules. The vast majority of PEGylating reagents react with free primary amino groups of the polypeptide. Most of these free amines are the epsilon amino group of lysine amino acid residues. Typical proteins possess a large number of lysines. Consequently, random attachment of multiple PEG molecules often occurs leading to loss of protein activity.
In addition, if the PEGylated protein is intended for therapeutic use, the multiple species mixture that results from the use of non-specific PEGylation leads to difficulties in the preparation of a product with reproducible and characterizable properties. This non-specific PEGylation makes it difficult to evaluate therapeutics and to establish efficacy and dosing information. The site selective PEGylation of such proteins could lead to reproducibly-modified materials that gain the desirable attributes of PEGylation without the loss of activity.
The need to reproducibly create complexes of two or more linked bioactive molecules or compounds also exists. In certain cases, the administration of multimeric complexes that contain more than one biologically active polypeptide or drug leads to synergistic benefits. For example, a complex containing two or more identical binding polypeptides may have substantially increased affinity for the ligand or active site to which it binds relative to the monomeric polypeptide. Alternatively, a complex comprised of (1) a bioactive protein that exerts its effect at a particular site in the body and (2) a molecule that can direct the complex to that specific site may be particularly beneficial.
A need also exists for hydrolytically-stable activated polymers which form linkages which are also hydrolytically stable. Otherwise, in certain cases, the reactive group can be rendered inactive before the desired reaction takes place or the conjugate formed after reaction has a short half life in aqueous media, such as blood or plasma.
For example, Zalipsky U.S. Pat. No. 5,122,614 describes that PEG molecules activated with an oxycarbonyl-N-dicarboximide functional group that can be attached under aqueous, basic conditions by a urethane linkage to the amine group of a polypeptide. Activated PEG-N-succinimide carbonate is said to form stable, hydrolysis-resistant urethane linkages with amine groups. The amine group is shown to more reactive at basic pHs of about 8.0 to 9.5, and reactivity falls off sharply at lower pHs. Hydrolysis of the uncoupled PEG derivative, however, also increases sharply at pHs of 8.0 to 9.5. Zalipsky avoids the problem of an increase in the rate of reaction of the uncoupled PEG derivative with water by using an excess of PEG derivative to bind to the protein. By using an excess of PEG derivative, sufficient reactive amino sites are bound to PEG to modify the protein before the PEG derivative becomes hydrolyzed and unreactive.
Zalipsky""s method is adequate for nonspecific attachment of the lysine fraction of a protein to a PEG derivative at one active site on the PEG. If the rate of hydrolysis of the PEG derivative is substantial, however, then it can be problematic to provide attachment at more than one active site on the PEG molecule, since a simple excess does not slow the rate of hydrolysis.
For example, a linear PEG with active sites at each end will attach to protein at one end but the reactive site at the other end can react with water to form a relatively nonreactive hydroxyl moiety instead of a PEG linking two protein groups. A similar problem arises if it is desired to couple a molecule to a surface by a PEG linking agent because the PEG is first attached to the surface or couples to the molecule, and the opposite end of the PEG derivative must remain active for a subsequent reaction. If hydrolysis is a problem, then the opposite end typically becomes inactivated.
Zalipsky U.S. Pat. No. 5,122,614 also describes several other PEG derivatives from prior patents. PEG-succinoyl-N-hydroxysuccinimide ester is said to form ester linkages that have limited stability in aqueous media. PEG-cyanuric chloride is said to be toxic and is non-specific for reaction with particular functional groups on a protein which can lead to protein inactivation. PEG-phenylcarbonate is said to produce toxic hydrophobic phenol residues that have an affinity for proteins. PEG activated with carbonyldiimidizole is said to be too slow in reacting with protein functional groups, requiring long reaction times to obtain sufficient modification of the protein.
Still other PEG derivatives have been proposed for attachment to functional groups other than the epsilon amino group of lysine. Maleimide, for example, is specific for cysteine sulfhydryl but the maleimide functionality is subject to hydrolysis.
Accordingly, a need exists for reagents and methods for reproducibly creating complexes whose parts are linked by nonantigenic, highly soluble, biologically inert molecules. The present invention satisfies the need for such complexes and provides related advantages. The present invention also satisfies the need for hydrolytically stable reagents that form hydrolytically stable conjugates.
The present invention relates to biologically-active conjugates containing a biologically-active molecule having a reactive thiol moiety and a non-peptidic polymer having an active sulfone moiety which forms a link with the reactive thiol moiety. The biologically-active molecule can be a synthetic, a naturally occurring, or a modified naturally occurring molecule. A molecule possessing the desired biological activity can be modified to contain a reactive thiol moiety.
Particularly useful biologically active molecules include the tumor necrosis factor (xe2x80x9cTNFxe2x80x9d) inhibitors, Interleukin-1 receptor antagonists (xe2x80x9cIL-1ra""s xe2x80x9d), CR1, exon six peptide of PDGF, and the Interleukin-2 (xe2x80x9cIL-2xe2x80x9d) inhibitors and receptors (xe2x80x9cIL-2rxe2x80x9d).
The polymer of the present invention contains at least one active sulfone moiety and has the formula Pxe2x80x94SO2xe2x80x94Cxe2x80x94C*xe2x80x94, where P is polymer and C* is a reactive site for linkage with thiol moieties. The link between the thiol and activated sulfone is at Cu and can be represented by the formula Pxe2x80x94SO2xe2x80x94Cxe2x80x94C*Sxe2x80x94R, where R is the biologically-active molecule. Useful activated sulfone moieties include, for example, vinyl sulfone and chloroethyl sulfone.
Various polymers can be activated for use in all embodiments of the present invention including water soluble polymers such as polyethylene glycol (xe2x80x9cPEGxe2x80x9d) and related hydrophilic polymers.
The present invention also provides methods of using sulfone-activated polymers to make the biologically-active conjugates discussed above. The method includes the steps of:
(a) reacting the biologically-active molecule having a reactive thiol moiety with a non-peptidic polymer having an active sulfone moiety to form a conjugate; and
(b) isolating the conjugate.
Pharmaceutical compositions containing the conjugates are also within the scope of the invention.
The present invention further relates to sulfone-activated polymers useful for coupling to a variety of molecules, compounds, and surfaces. The activated sulfone moiety is the same as discussed above. Particularly useful activated polymers include bifunctional PEG derivatives activated with a sulfone moiety at one site on the PEG molecule and an NHS-ester or a maleimide functionality at another site.
Further included in the present invention are substantially purified biologically-active compounds having the formula R1xe2x80x94Xxe2x80x94R2, called a xe2x80x9cdumbbellxe2x80x9d where at least one of R1 or R2 is a biologically-active molecule which retains its biological activity when part of the compound. The biologically-active molecule has a reactive thiol moiety which forms a link with a Michael acceptor group on a non-peptidic polymer. Biologically-active molecules suitable for use in the present invention include those mentioned above. Useful Michael acceptor groups include, for example, vinyl sulfone and maleimide. Polymers which can be activated with Michael acceptor functional groups include the water soluble polymers mentioned above.
R1 and R2 can be the same or different moieties. When the R groups are the same, the compound is a homodumbbell; when the R groups are different, the compound is a heterodumbbell. Particularly useful homodumbbells include, for example, PEG-linked TNF inhibitors and PEG-linked IL-1ra""s. Useful heterodumbbells include, for example, those formed from IL-2r-xcex1 and IL-2r-xcex2, heterodumbbells which inhibit the classical pathway of the complement system, and heterodumbbells formed from IL-1ra and exon 6 of PDGF.
Methods of making the dumbbell compounds are within the scope of the invention. The methods of making a dumbbell, R1xe2x80x94Xxe2x80x94R2, include the steps of:
(a) reacting X with R1 and R2 to form R1xe2x80x94Xxe2x80x94R2; and
(b) purifying R1xe2x80x94Xxe2x80x94R2.
Step (a) in the above methods of making dumbbells can further include the following steps:
protecting one reactive group of X to form a protected group on X;
reacting X having a protected group with R1 to form R1xe2x80x94X;
deprotecting the protected group on X; and
reacting R1xe2x80x94X with R2 to form R1xe2x80x94Xxe2x80x94R2.
Alternatively or in addition, step (a) can further include the following steps:
reacting an excess of X with R1 to form R1xe2x80x94X; and
reacting R1xe2x80x94X with R2 to form R1xe2x80x94Xxe2x80x94R2.
Pharmaceutical compositions containing the substantially purified compounds R1xe2x80x94Xxe2x80x94R2 are also within the scope of the invention.
The present invention provides biologically-active conjugates containing (1) a biologically-active molecule having a reactive thiol moiety, and (2) a non-peptidic polymer having an active sulfone moiety which forms a linkage with the thiol moiety of the biologically-active molecule.
A xe2x80x9cconjugatexe2x80x9d means a complex that is formed by joining a biologically-active molecule, having an active thiol moiety, to a non-peptidic polymer, having an active sulfone moiety, via a linkage between the thiol and sulfone. As stated above, the conjugates of the present invention are biologically active.
xe2x80x9cBiologically activexe2x80x9d means capable of exerting a biological effect, in vitro or in vivo. A biologically active molecule includes, but is not limited to, any compound that can induce a biological effect on interaction with a natural biological molecule or on a biological system such as a cell or organism. Ways of demonstrating biological activity include in-vitro bioassays, many of which are well known in the art. For example, one can measure the biological activity of tumor necrosis factor (xe2x80x9cTNFxe2x80x9d) inhibitors by determining if the inhibitors bind to TNF or if the inhibitors block TNF-mediated lysis of certain cells. The latter bioassay is set forth in published European Patent Application No. 90113673.9, which is specifically incorporated herein by reference.
Biologically-active molecules include, but are not limited to, pharmaceuticals, vitamins, nutrients, nucleic acids, amino acids, polypeptides, enzyme co-factors, steroids, carbohydrates, organic species such as heparin, metal containing agents, receptor agonists, receptor antagonists, binding proteins, receptors or portions of receptors, extracellular matrix proteins, cell surface molecules, antigens, haptens, targeting groups, and chelating agents. All references to receptors include all forms of the receptor whenever more than a single form exists.
xe2x80x9cPolypeptidesxe2x80x9d and xe2x80x9cproteinsxe2x80x9d are used herein synonymously and mean any compound that is substantially proteinaceous in nature. However, a polypeptidic group may contain some non-peptidic elements. For example, glycosylated polypeptides or synthetically modified proteins are included within the definition. xe2x80x9cTargeting groupsxe2x80x9d can direct a compound to a location in a biological system. Binding proteins and receptors can be described by their affinity for a certain ligand.
Many polypeptides useful in the present invention are set forth in published PCT Publication No. WO 92/16221, specifically incorporated herein by reference. These proteins are well known in the art. Particularly useful polypeptides are the TNF binding proteins, also called TNF inhibitors. A xe2x80x9cTNF binding proteinxe2x80x9d is defined herein to mean a protein that binds TNF.
One TNF binding protein (xe2x80x9cTNFbpxe2x80x9d) is the extracellular portion of the p55 TNF receptor or the TNF receptor I. In vivo, the extracellular portion of the receptor is shed and circulates in the bloodstream as a 30 kDa glycosylated protein which binds to TNF. This binding protein is also referred to TNFbp-I or the 30 kDa TNFbp. The purification and amino acid and nucleic acid sequences of this TNF binding protein are set forth in published European Patent Application No. 90 113 673.9, which is incorporated herein by reference.
This published reference also teaches the recombinant production of glycosylated and deglycosylated forms of this TNF inhibitor. Although the actual molecular weight of the deglycosylated form of this inhibitor is approximately 18 kDa, the term xe2x80x9c30 kDa TNF inhibitorxe2x80x9d includes the glycosylated and deglycosylated forms.
As used herein, the terms xe2x80x9cnaturally-occurring,xe2x80x9d xe2x80x9cnative,xe2x80x9d and xe2x80x9cwild-typexe2x80x9d are synonymous.
European Patent Application No. 90 113 673.9, incorporated herein by reference, also sets forth the purification and amino acid and nucleic acid sequences of another TNF inhibitor, called the 40 kDa TNF inhibitor. Also called TNFbp-II, this inhibitor, in its naturally-occurring form, is the glycosylated extracellular portion of the p75 or p85 TNF receptor. European Patent Application No. 90 112 673.9 also teaches the recombinant production of the glycosylated and deglycosylated forms of this xe2x80x9c40 kDaxe2x80x9d inhibitor. The nucleic and amino acid sequences of the native 40 kDa TNF inhibitor are set forth in this published reference. Although the molecular weight of the deglycosylated form is not 40 kDa, both the glycosylated and deglycosylated forms of this TNFbp are referred to as xe2x80x9c40 kDa TNF inhibitor.xe2x80x9d
European Patent Application No. 90 112 673.9, incorporated herein by reference, further teaches the recombinant production of two TNF inhibitors which are portions of the full length xe2x80x9c40 kDaxe2x80x9d binding protein. These two truncates are called the xe2x80x9cxcex9451xe2x80x9d and xe2x80x9cxcex9453xe2x80x9d TNF inhibitors. The amino acid and nucleic acid sequences of the xcex9451 and xcex9453 inhibitors are set forth in this published reference.
Other particularly useful polypeptides include the interleukin-1 receptor antagonists (xe2x80x9cIL-1ra""sxe2x80x9d), as described in U.S. Pat. No. 5,075,222, incorporated herein by reference, insulin-like growth factor binding proteins (xe2x80x9cIGFbpsxe2x80x9d), CTLA4, and exon six of platelet derived growth factor (xe2x80x9cPDGFxe2x80x9d), glial derived neurotrophic factor (xe2x80x9cGDNFxe2x80x9d), ciliary neurotrophic factor (xe2x80x9cCNTFxe2x80x9d), interleukin-4 receptor (xe2x80x9cIL-4r), and inhibitors, and interleukin-1 receptor (xe2x80x9cIL-2rxe2x80x9d). The nucleic acid encoding the naturally occurring IL-1ra and a method for expressing the protein in E. Coli. are set forth in U.S. Pat. No. 5,075,222 of Hannum et al.
Characteristics of the IL-2 receptors and CR1, the nucleic acids encoding them, and methods for their production are discussed in published PCT Publication No. WO 92/16221, specifically incorporated herein by reference.
The biologically-active molecules linked to polymers in the conjugates of the present invention have a reactive thiol moiety prior to forming the linkage. A xe2x80x9creactive thiol moietyxe2x80x9d means a xe2x80x94SH group capable of reacting with the activated polymers as described herein.
An example of a reactive thiol is the xe2x80x94SH of the amino acid cysteine. Many proteins do not have free cysteines (cysteines not involved in disulfide bonding) or any other reactive thiol group. In addition, the cysteine thiol may not be appropriate for linkage to the polymer because the thiol is necessary for biological activity. In addition, proteins must be folded into a certain conformation for activity. In the active conformation, a cysteine can be inaccessible for reaction with sulfone because it is buried in the interior of the protein. Moreover, even an accessible cysteine thiol which is not necessary for activity can be an inappropriate site to form a linkage to the polymer. Amino acids not essential for activity are termed xe2x80x9cnonessential.xe2x80x9d Nonessential cysteines can be inappropriate conjugation sites because the cysteine""s position relative to the active site results in the polypeptide becoming inactive after conjugation to polymer. Like proteins, many other biologically-active molecules have reactive thiols which, for reasons similar to those recited above, are not suitable for conjugation to the polymer or contain no reactive thiol groups.
Accordingly, the present invention contemplates the introduction of reactive thiol groups into a biologically-active molecule when necessary or desirable. Thiol groups can also be introduced into an inactive molecule to form a biologically-active molecule as long as the thiol-sulfone link does not destroy the desired activity.
Reactive thiol groups can be introduced by chemical means well known in the art. Chemical modification can be used with polypeptides or non-peptidic molecules and includes the introduction of thiol alone or as part of a larger group, for example a cysteine residue, into the molecule. An example of chemically introducing thiol is set forth in Jue, R. et al., Biochemistry, 17, pp. 5399-5406 (1978). One can also generate a free cysteine in a polypeptide by chemically reducing cystine with, for example, DTT.
Polypeptides which are modified to contain an amino acid residue in a position where one was not present in the native protein before modification is called a xe2x80x9cmutein.xe2x80x9d To create cysteine muteins, a nonessential amino acid can be substituted with a cysteine or a cysteine residue can be added to the polypeptide. Potential sites for introduction of a non-native cysteine include glycosylation sites and the N or C terminus of the polypeptide. The mutation of lysine to cysteine is also appropriate because lysine residues are often found on the surface of a protein in its active conformation. In addition, one skilled in the art can use any information known about the binding or active site of the polypeptide in the selection of possible mutation sites.
One skilled in the art can also use well known recombinant DNA techniques to create cysteine muteins. One can alter the nucleic acid encoding the native polypeptide to encode the mutein by standard site directed mutagenesis. Examples of standard mutagenesis techniques are set forth in Kunkel, T. A., Proc. Nat. Acad. Sci., Vol. 82, pp. 488-492 (1985) and Kunkel, T. A. et al., Methods Enzymol., Vol. 154, pp. 367-382 (1987), both of which are incorporated herein by reference. Alternatively, one can chemically synthesize the nucleic acid encoding the mutein by techniques well known in the art. DNA synthesizing machines can be used and are available, for example, from Applied Biosystems (Foster City, Calif.). The nucleic acid encoding the desired mutein can be expressed in a variety of expression systems, including animal, insect, and bacterial systems.
When the mutein is recombinantly produced in a bacterial expression system, the following steps are performed:
1) The nucleic acid encoding the desired mutein is created by site directed mutagenesis of the nucleic acid encoding the native polypeptide;
2) The nucleic acid encoding the desired mutein is expressed in a bacterial expression system;
3) The mutein is isolated from the bacteria and purified;
4) If not folded properly, the mutein is refolded in the presence of cysteine or another sulphydryl containing compound;
5) The refolded mutein is isolated and purified;
6) The purified and refolded target mutein is treated with a mild reducing agent;
7) The reaction mixture is dialyzed in the absence of oxygen.
As discussed below, the mutein can be isolated from the reaction mixture prior to conjugation with polymer but need not be. A reducing agent particularly useful in step 6 is dithiothreitol (xe2x80x9cDTTxe2x80x9d) or Tris-(carboxyethylphosphine) (xe2x80x9cTCEPxe2x80x9d). TCEP is useful because it does not have to be removed before conjugation with a thiol-specific PEG reagent. See Burns, J. A. et al., J. Org. Chem., Vol.56, No. 8, pp. 2648-2650 (1991).
After creation of the desired mutein, one skilled in the art can bioassay the mutein and compare activity of the mutein relative to the native polypeptide. As more fully discussed below, even if the relative activity of the mutein is diminished, the conjugate formed from the mutein can be particularly useful. For example, the conjugate can have increased solubility, reduced antigenicity or immunogenicity, or reduced clearance time in a biological system relative to the unconjugated molecule. Such improvements in the pharmacokinetic performance of the biologically-active molecule can increase the molecule""s value in various therapeutic applications. Increased solubility can also improve the value of the molecule for in-vitro diagnostic applications.
Table 1 lists muteins of IL-1ra that have been produced. The preparation and purification of IL-1ra muteins are set forth in published PCT Patent Publication No. WO 92/16221, specifically incorporated herein by reference. The residue numbering is based upon the sequence set forth in that published application with xe2x80x9c0xe2x80x9d denoting addition of an amino acid at the N-terminus; xe2x80x9ccxe2x80x9d referring to cysteine and xe2x80x9csxe2x80x9d referring to serine. For example, xe2x80x9cc0s116xe2x80x9d means a cysteine was inserted at the N terminus and a serine was inserted at position 116. Native IL-1ra has free cysteine residues at positions 66, 69, 116 and 122.
Table 2 shows muteins of the 30 kDa TNF inhibitor which have also been prepared. The native 30 kDa TNF inhibitor, unlike IL-1ra, does not have any free cysteine residues. These muteins have been prepared as set forth in published PCT Publication No. WO 92/16221, specifically incorporated herein by reference, and the numbering is based upon the amino acid sequence set forth therein.
The muteins and other polypeptides of the present invention include allelic variations in the protein sequence and substantially equivalent proteins. xe2x80x9cSubstantially equivalent,xe2x80x9d means possessing a very high degree of amino acid residue homology (See generally, M. Dayhoff, Atlas of Protein Sequence and Structure, vol. 5, p. 124 (1972), National Biochemical Research Foundation, Washington, D.C., specifically incorporated herein by references) as well as possessing comparable biological activity. Also included within the scope of this invention are truncated forms of the native polypeptide or mutein that substantially retain the biological activity of the native polypeptide or mutein.
The conjugates of the present invention contain, in addition to biologically-active molecules having reactive thiol moieties, non-peptidic polymeric derivatives having active sulfone moieties. xe2x80x9cNon-peptidicxe2x80x9d means having less than 50% by weight of xcex1 amino acid residues.
The polymer portion of the polymeric derivative can be, for example, polyethylene glycol (xe2x80x9cPEGxe2x80x9d), polypropylene glycol (xe2x80x9cPPGxe2x80x9d), polyoxyethylated glycerol (xe2x80x9cPOGxe2x80x9d) and other polyoxyethylated polyols, polyvinyl alcohol (xe2x80x9cPVA) and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glucose. The polymer can be a homopolymer, a random or block copolymer, a terpolymer based on the monomers listed above, straight chain or branched, substituted or unsubstituted as long as it has at least one active sulfone moiety. The polymeric portion can be of any length or molecular weight but these characteristics can affect the biological properties. Polymer average molecular weights particularly useful for decreasing clearance rates in pharmaceutical applications are in the range of 2,000 to 35,000 daltons. In addition, if two groups are linked to the polymer, one at each end, the length of the polymer can impact upon the effective distance, and other spatial relationships, between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or confer the desired biological activity. If the polymer is a straight chain PEG, particularly useful lengths of polymers, represented by (Z)n, where Z is the monomeric unit of the polymer, include n having a range of 50-500. In certain embodiments of the present invention, n is greater than 6 and preferably greater than 10.
Monomethoxy polyethylene glycol is designated here as mPEG. The term xe2x80x9cPEGxe2x80x9d means any of several condensation polymers of ethylene glycol. PEG is also known as polyoxyethylene, polyethylene oxide, polyglycol, and polyether glycol. PEG can also be prepared as copolymers of ethylene oxide and many other monomers. For many biological or biotechnical applications, substantially linear, straight-chain vinyl sulfone activated PEG will be used which is substantially unsubstituted except for the vinyl sulfone.
PEG is useful in biological applications for several reasons. PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze, and is nontoxic. PEGylation can improve pharmacokinetic performance of a molecule by increasing the molecule""s apparent molecular weight. The increased apparent molecular weight reduces the rate of clearance from the body following subcutaneous or systemic administration. In many cases, PEGylation can decrease antigenicity and immunogenicity. In addition, PEGylation can increase the solubility of a biologically-active molecule.
The polymeric derivatives of the present invention have active sulfone moieties. xe2x80x9cActive sulfonexe2x80x9d means a sulfone group to which a two carbon group is bonded having a reactive site for thiol-specific coupling on the second carbon from the sulfone group at about pH 9 or less. Examples of active sulfones include, but are not limited to, vinyl sulfone and activated ethyl sulfone. An example of an active ethyl sulfone is xe2x80x94SO2xe2x80x94CH2xe2x80x94CH2xe2x80x94Z where Z is halogen or another leaving group capable of substitution by thiol to form the sulfone-thiol linkage xe2x80x94SO2xe2x80x94CH2xe2x80x94CH2xe2x80x94R, where R represents a biologically active molecule. The sulfone-activated polymer can be further substituted as long as the thiol-specific reactivity at the second carbon is maintained at about pH 9 or less.
The sulfone-activated polymers of the present invention can be synthesized in at least four steps. Briefly, the first step is to increase the reactivity of a site on the polymer, typically an end group, by, for example, activation or substitution. The second step is to link sulfur directly to a carbon atom in the polymer in a form that can be converted to an ethyl sulfone or ethyl sulfone derivative having similar reactive properties. In the third step, the sulfur is oxidized to sulfone. In the fourth step, the second carbon from the sulfone group is activated.
The synthesis of a sulfone-activated polymer is described in more detail below using the synthesis of a sulfone-activated PEG as an example. The first step is the hydroxyl activation of an hydroxyl moiety in the PEG. The term xe2x80x9chydroxyl activationxe2x80x9d should be interpreted herein to mean substitution as well as esterification and other methods of hydroxyl activation. Typically, in hydroxyl activation, an acid or an acid derivative such as an acid halide is reacted with the PEG to form a reactive ester in which the PEG and the acid moiety are linked through the ester linkage. The acid moiety generally is more reactive than the hydroxyl moiety. Typical esters are the sulfonate, carboxylate, and phosphate esters.
Sulfonyl acid halides that are suitable for use in the invention include, for example, methanesulfonyl chloride (also known as mesyl chloride) and p-toluene-sulfonyl chloride (also known as tosyl chloride). Methanesulfonate esters are sometimes referred to as mesylates. Toluenesulfonate esters are sometimes referred to as tosylates.
In a substitution type of hydroxyl activation, the entire hydroxyl group on the PEG is substituted by a more reactive moiety, typically a halide. For example, thionyl chloride, can be reacted with PEG to form a more reactive chlorine substituted PEG.
Thus, when PEG is the starting material, the typical reaction product of the first step is an ester or halide-substituted PEG.
In the second step, the ester or halide is substituted by an alcohol which contains a reactive thiol attached to an ethyl group, a thioethanol moiety. Thioethanol is an example of a suitable alcohol. In this step, the sulfur in the thiol is bonded directly to a carbon on the polymer.
Next, in the third step, the sulfur is oxidized to sulfone. Useful oxiding agents include, for example, hydrogen peroxide, sodium perborate, or peroxy acids.
In the fourth step, the hydroxyl moiety of the alcohol used in step two is activated. This step is similar to the first step in the reaction sequence. Substitution typically is with halide to form a haloethyl sulfone or a derivative thereof having a reactive site on the second carbon removed from the sulfone moiety. Typically, the second carbon on the ethyl group will be activated by a chloride or bromide halogen. Hydroxyl activation should provide a site of similar reactivity, such as the sulfonate ester. Suitable reactants are, for example, the acids, acid halides, and others previously mentioned in discussing the first step in the reaction. Thionyl chloride is particularly useful for substitution of the hydroxyl group with the chlorine atom.
The resulting polymeric activated ethyl sulfone is stable, isolatable, and suitable for thiol-selective coupling reactions. PEG chloroethyl sulfone is stable in water at a pH of about 7 or less, but nevertheless can be used to advantage for thiol-selective coupling reactions at conditions of basic pH up to at least about pH 9. At a pH of above about 9, the thiol selectivity is diminished and the sulfone moiety becomes somewhat more reactive with amino groups. The linkage formed upon reaction with thiol is also hydrolytically stable.
In a fifth step that can be added to the synthesis, the activated ethyl sulfone is reacted with a base to from PEG vinyl sulfone or one of its active derivatives for thiol-selective coupling. Suitable bases include, for example, sodium hydroxide or triethylamine. Like activated ethyl sulfones, vinyl sulfone is hydrolytically stable, isolatable, thiol-selective, and forms hydrolytically-stable linkages upon reaction with thiol.
As used herein, xe2x80x9chydrolytically stablexe2x80x9d means that the linkage between the polymer and the sulfone moiety and between the sulfone-thiol after conjugation does not react with water at a pH of less than about 11 for at least three days. Hydrolytic stability is desirable because, if the rate of hydrolysis is significant, the polymer can be deactivated before the reaction between polymer and the thiol of the biologically-active molecule takes place.
As mentioned above, for example, a linear PEG with active sites at each end will attach to a protein at one end, but, if the rate of hydrolysis is significant, will react with water at the other end to become capped with a relatively nonreactive hydroxyl moiety, rather than forming a xe2x80x9cdumbbellxe2x80x9d molecular structure with attached proteins or other desirable groups on each end. A similar problem arises when coupling a molecule to a surface by a PEG linking agent because the PEG is first attached to the surface or couples to the molecule, and the opposite end of the PEG derivative must remain active for a subsequent reaction. If hydrolysis is a problem, then the opposite end typically becomes inactivated.
Alternatively, the sulfone-activated derivatives can be prepared by attaching a linking agent having a sulfone moiety to a PEG (or other polymer) activated with a different functional group. For example, an amino activated PEG can be reacted under favorable conditions of pH of about 9 or less with a small molecule that has a succinimidyl active ester moiety at one terminus and vinyl sulfone at the other terminus. The amino-activated PEG forms a stable linkage with the succinimidyl ester. The resulting PEG is activated with the vinyl sulfone at the terminus and is hydrolytically stable: PEGxe2x80x94NHxe2x80x94OCxe2x80x94CH2xe2x80x94CH2xe2x80x94SO2CHxe2x95x90CH2.
A similar activated PEG can be achieved by reacting an amine-reactive PEG such as succinimidyl active ester PEG, PEGxe2x80x94CO2xe2x80x94NHS, with a small molecule that has an amine moiety at one terminus and a vinyl sulfone moiety at the other terminus.
PEG chloroethyl sulfone and PEG vinyl sulfone were prepared as set forth in Example 1. Thiol-selective reactivity of PEG vinyl sulfone and chloroethyl sulfone is shown in Example 2. Hydrolytic stability of the polymer-sulfone linkage of two compounds is shown in Example 3. Hydrolytic stability of the linkage between thiol and sulfone is shown in Example 16.
When the polymer does not have an hydoxyl moiety, one can first be added by chemical methods well known in the art before carrying out the steps described above.
The activated polymeric derivatives of the present invention can have more than one reactive group. The derivatives can be monofunctional, bifunctional, or multifunctional. The reactive groups may be the same (homofunctional) or different (heterofunctional) as long as there is at least one active sulfone moiety.
Two particularly useful homobifunctional derivatives are PEG-bis-chlorosulfone and PEG-bis-vinyl sulfone. One skilled in the art can synthesize those molecules using PEG having hydroxyl moieties at each end as a starting material and following the general method set forth above.
Heterobifunctional derivatives can also be synthesized. Two particularly useful heterobifunctional derivatives include, for example, a linear PEG with either a vinyl sulfone or a maleimide at one end and an N-hydroxysuccinimide ester (xe2x80x9cNHS-esterxe2x80x9d) at the other end. The NHS-ester is amine-specific. PEG having an NHS-ester at one end and an activated sulfone moiety at the other can be attached to both lysine and cysteine residues. A stable amine linkage can be achieved, leaving the hydrolytically-stable unreacted sulfone available for subsequent reaction with thiol. Those two heterobifunctional PEG derivatives have been synthesized as described in Examples 5 and 6. If the maleimide NHS-ester heterobifunctional reagent is made using straight-chain PEG, represented by (Z)n, where Z is the monomeric unit, n is greater than 6 and preferably greater than 10.
Other active groups for heterofunctional sulfone-activated PEGs can be selected from among a wide variety of compounds. For biological and biotechnical applications, the substituents would typically be selected from reactive moieties typically used in PEG chemistry to activate PEG such as the aldehydes, trifluoroethylsulfonate (sometimes called tresylate), n-hydroxylsuccinimide ester, cyanuric chloride, cyanuric fluoride, acyl azide, succinate, the p-diazo benzyl group, the 3-(p-diazophenyloxy)-2-hydroxy propyloxy group, and others.
Examples of active moieties other than sulfone are shown in Davis et al. U.S. Pat. No. 4,179,337; Lee et al. U.S. Pat. Nos. 4,296,097 and 4,430,260; Iwasaki et al. 4,670,417; Katre et al. U.S. Pat. Nos. 4,766,106; 4,917,888; and 4,931,544; Nadagawa et al. U.S. Pat. No. 4,791,192; Nitecki et al. U.S. Pat. No. 4,902,502 and 5,089,261; Saifer U.S. Pat. No. 5,080,891; Zalipsky U.S. Pat. No. 5,122,614; Shadle et al. U.S. Pat. No. 5,153, 265; Rhee et al. U.S. Pat. No. 5,162,430; European Patent Application Publication No. 0 247 860; and PCT International Application Nos. US86/01252; GB89/01261; GB89/01262; GB89/01263; US90/03252; US90/06843; US91/06103; US92/00432; and US92/02047, the contents of which are incorporated herein by reference.
An example of a trifunctional derivative is a glycerol backbone to which three vinyl sulfone PEG moieties are attached. This molecule can be represented by the formula: 
This derivative was prepared as described in Example 12.
Another example of a multifunctional derivative is the xe2x80x9cstarxe2x80x9d molecule. Star molecules are generally described in Merrill U.S. Pat. No. 5,171,264, incorporated herein by reference. Star molecules have a core structure to which multiple PEG chains or xe2x80x9carmsxe2x80x9d are attached. The sulfone moieties can be used to provide an active, functional group on the end of the PEG chain extending from the core and as a linker for joining a functional group or other moiety to the star molecule arms.
It should be apparent to the skilled artisan that the activated polymers discussed above could be used to carry a wide variety of substituents and combinations of substituents.
As stated above, the conjugates of the present invention are formed by reacting thiol-containing biologically-active molecules with sulfone-activated polymers. The linkage between the thiol reactive group and the sulfone-activated polymer is a covalent bond.
A general method for preparing the conjugates of the present invention includes the following steps:
(1) Choose the desired biologically-active molecule and determine if the molecule possesses a free thiol group by means well known in the art. See, for example, Allen, G., xe2x80x9cSequencing of Proteins and Peptides,xe2x80x9d pp. 153-54, in Laboratory Techniques in Biochemistry and Molecular Biology, Work, T. S., and Burdon, R. H., eds. (1972), incorporated herein by reference. If the molecule has a free thiol, proceed to step 3. If the molecule has no free thiol, proceed to step 2.
(2) If no free thiol exists in the molecule, add thiol as discussed above. After adding thiol, perform a bioassay to determine if the desired biological activity or a portion of the biological activity is retained.
(3) Synthesize the desired sulfone-activated polymer as discussed above.
(4) React the activated polymer with the molecule having a free thiol.
(5) Isolate the reaction product using chromatographic techniques well known in the art. For protein conjugates, see, for example, Scopes, R., Protein Purification, Cantor, C. R. ed., Springer-Verlag, New York (1982). For nonprotein molecules, see, for example, Still, W. C. et al., J. Org. Chem., 43, pp.2923-2925 (1978). If no conjugate forms, add thiol to another location on the biologically-active molecule and repeat steps (4) and (5).
(6) Determine biological activity of the conjugate formed using the relevant bioassay.
One skilled in the art can add or delete certain steps. For example, one skilled in the art might not assay bioactivity in step 2 or might presume biological activity after PEGylation based upon previous experiments. The skilled artisan can also add the step of varying the size, length, or molecular weight of the linker to optimize or confer biological activity.
Several conjugates have been prepared. The 30 kDa TNFbp c105 mutein described above was conjugated with PEG vinyl sulfone as described in Example 10. Example 8 shows that native IL-1ra, which contains four free cysteines, reacted under similar conditions. The c84 IL-1ra mutein also reacted well. Example 13 shows the conjugation of three 30 kDa TNF inhibitor muteins to three PEG chains bonded to a glycerol backbone.
The conjugates of the present invention can be used for a variety of purposes including, but not limited to, in-vitro diagnostic assays and the preparation of pharmaceutical compositions. Many of the conjugates of the present invention have at least one of the following characteristics relative to the unconjugated molecule:
(1) increased solubility in aqueous solution;
(2) reduced antigenicity or immunogenicity;
(3) reduced rate of clearance following subcutaneous or systemic administration due to increased apparent molecular weight.
Pharmaceutical preparations of conjugates containing IL-1ra are particularly useful. IL-1ra, alone or in combination with the 30 kDa TNF binding protein, can be used to treat arthritis, inflammatory bowel disease, septic shock, ischemia injury, reperfusion injury, osteoporosis, asthma, insulin diabetes, myelogenous and other leukemias, psoriasis, adult respiratory distress syndrome, cachexia/anorexia, and pulmonary fibrosis.
Conjugates containing TNF binding proteins (xe2x80x9cTNFbpsxe2x80x9d) are also particularly useful. Such conjugates can be used to treat TNF-mediated diseases such as adult respiratory distress syndrome, pulmonary fibrosis, arthritis, septic shock, inflammatory bowel disease, multiple sclerosis, graft rejection and hemorrhagic trauma.
The biologically active conjugates of the present invention can further include non-biologically active moieties.
The present invention also includes substantially purified compounds having the formula R1xe2x80x94Xxe2x80x94R2, where at least one of R1 and R2 is a biologically-active molecule having a reactive thiol moiety which forms a covalent bond with X, a Michael acceptor-activated polymer. In the present invention, the biological activity of R1xe2x80x94Xxe2x80x94R2 retains the biological activity of R1 or R2. Molecules having the formula R1xe2x80x94Xxe2x80x94R2 are referred to herein as xe2x80x9cdumbbellxe2x80x9d molecules.
As stated above, the compounds of the present invention are substantially purified. xe2x80x9cSubstantially purifiedxe2x80x9d as used herein means a xe2x80x9chomogenous composition.xe2x80x9d A homogenous composition contains molecules of R1xe2x80x94Xxe2x80x94R2 and is substantially free from compounds that (1) deviate in the composition of R1 or R2, or (2) are linked together by more than one activated polymer. The homogeneous composition can contain molecules of R1xe2x80x94Xxe2x80x94R2 which differ in the length of X. For straight-chain polymers, represented by (Z)n, where Z is the monomeric unit, n is greater than 6 and preferably greater than 10. To have a homogeneous composition, R1 and R2 need not be attached to X at the same location on X or on the same location on either R group.
X is a non-peptidic polymer having a first reactive group and a second reactive group. A xe2x80x9creactive groupxe2x80x9d is a group capable of reacting with R. At least one reactive group on X is a Michael-type acceptor. The terms xe2x80x9creactive groupxe2x80x9d and xe2x80x9cfunctional groupxe2x80x9d are used herein synonymously. The terms xe2x80x9cMichael acceptorxe2x80x9d and xe2x80x9cMichael-type acceptorxe2x80x9d are also used herein synonymously. Polymers suitable for use in the present invention are also discussed above and include, for example, PEG, POG, and PVA.
xe2x80x9cMichael acceptorsxe2x80x9d are functional groups susceptible to Michael addition. xe2x80x9cMichael additionxe2x80x9d involves a nucleophilic attack on an electrophilic center which is adjacent to a pi system, having an electronegative atom. Examples of pi systems having an electronegative atom include sulfoxide, sulfonyl, carbonyl and heterocyclic aromatics. The nucleophile adds to the electrophilic center.
Michael acceptors can be represented by the formula: 
where E is an electronegative atom. Addition takes place at the 4 position to form the following: 
where Nu represents the nucleophile now bonded to the atom at position 4. Michael acceptor functional groups include, but are not limited to, maleimide and vinyl sulfone. The activated polymer from which a dumbbell is formed can, but need not, contain a vinyl sulfone species of Michael acceptor.
Activated polymers of the present invention include PEG having two or more Michael acceptor groups, including for example, PEG-bis-vinyl sulfone and PEG-bis-maleimide. PEG-bis-vinyl sulfone has been prepared as described in Examples 7. PEG-bis-maleimide has been prepared as described in PCT Publication No. WO 92/16221, incorporated herein by reference.
At least one of R1 and R2 is biologically active prior to coupling to X or to Xxe2x80x94R. xe2x80x9cBiologically activexe2x80x9d has the same definition recited above. As stated above, biologically active molecules include, but are not limited to, binding proteins and targeting groups.
Both R1 and R2 can be biologically active but need not be. In some cases, if R1 and R2 have an affinity for the same ligand, the dumbbell can have a greater affinity for that ligand than either R1 or R2 alone. Published PCT Publication No. WO 92/16221 shows that the homodumbbell containing two molecules of 30 kDa TNFbp linked by a PEG polymer is better at inhibiting cytotoxicity of TNFs in in-vitro assays than the 30 kDa molecule alone. In certain cases, R1 can be a molecule which directs the compound R1xe2x80x94Xxe2x80x94R2 to a certain location in a biological system and R2 can have an affinity for a ligand in that location.
Alternatively, only one of R1 and R2 can be biologically active in the compound R1xe2x80x94Xxe2x80x94R2. The nonbiologically-active group can be a surface or any other biologically-inert molecule or compound.
In the present invention, the biologically active R group has a reactive thiol moiety. The biologically active R group can be a synthetic molecule. As used herein, the term xe2x80x9csynthetic moleculexe2x80x9d means a molecule to which a reactive thiol moiety has been added. Synthetic molecules include, for example, muteins containing a non-native cysteine. The thiol moiety reacts with a Michael-type acceptor of the polymer to form a covalent bond.
After formation of this covalent bond, the biologically-active molecule retains its biological activity. The R group xe2x80x9cretains its biological activityxe2x80x9d within the meaning of the invention if, after reaction with activated polymer, it has at least one tenth of the biological activity it had before reaction with polymer, preferably at least 40%, and more preferably at least 60%.
A general method for producing dumbbells follows:
(1) Choose an R group possessing the desired biological activity, for example, a protein such as tumor necrosis factor binding protein (TNFbp).
(2) Measure activity using the relevant bioassay.
(3) Determine the number of free sulfhydryl groups, for example, cysteine residues not involved in disulfide bonding, using generally known methods in the art. One such method is described in Allen, G., xe2x80x9cSequencing of proteins and peptides,xe2x80x9d pp. 153-54, in Laboratory Techniques in Biochemistry and Molecular Biology, Work, T. S., and Burdon, R. H., eds. (1972). If there are no free cysteines, proceed to step 4(a). If there is one free cysteine, or only one accessible to the PEGylation reagent, proceed to the reaction step in 4(c). If the protein has more than one free cysteine, go to step 5.
(4) When R is polypeptide and no free cysteines exist:
(a) Create a mutein by inserting a cysteine or replacing a non-cysteine residue with a cysteine. Useful mutation sites include the N or C terminal ends of the protein, glycosylation sites, or lysine residues. Muteins can be routinely made, as stated above, by chemical synthesis or recombinant technology. Alternatively, chemically add a thiol moiety.
(b) Measure activity and compare that activity with the activity measured in step 2.
(c) If the mutein retains the activity measured in step 2, react the mutein with a polymer, such as PEG, having a single sulfhydryl-preferred reactive group. If the mutein bonds to the mono-reactive PEG (becomes PEGylated), measure activity and compare that activity with the activity measured in step 2. If the PEGylated mutein retains the activity measured in step 2, react the unPEGylated mutein with a PEG having two thiol-specific Michael Acceptors, such as bis-maleimide, to create dumbbell molecules. Repeat the bioassay to confirm that the dumbbells retain biological activity.
If one skilled in the art desires that R1 and R2 be different, the bis-reactive polymeric group can be reacted in series with R1 and then R2. Prior to reacting polymer with R1, one of the two functional groups of the polymer is blocked or protected by means well known in the chemical arts to form a protected group on X. See, for example, Greene, T. W. et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Inc. (1991), incorporated herein by reference. In this context, xe2x80x9cprotectedxe2x80x9d means the functional group is not available for reaction. When X having a protected group is reacted with R1, R1xe2x80x94X, and not R1xe2x80x94Xxe2x80x94R1, is formed. After R1xe2x80x94X is formed, the blocking or protecting group is removed prior to reaction with R2. xe2x80x9cDeprotectedxe2x80x9d means the protective group is removed or the functional group is otherwise made available for reaction.
Alternatively, heterodumbbells can be formed by reacting R1 with an excess of the bis-activated polymer to force R1xe2x80x94X formation. After reaction, R1xe2x80x94X is separated from the reaction mixture using chromatographic techniques well known in the art, including, for example, ion exchange chromatography. R1xe2x80x94X is then reacted with R2 to form R1xe2x80x94Xxe2x80x94R2.
(d) If the mutein created in step 4(a) or the PEGylated mutein formed in step 4(c) does not substantially retain biological activity, start with the native protein, create a different mutein, and repeat steps 4(b) and 4(c). In addition, the length or molecular weight of the polymer X can be changed to optimize or confer biological activity.
(5) For proteins with more than one free cysteine, monoPEGylate, bioassay, and react with the bifunctional PEGylation reagent. If higher-ordered structures are formed, i.e. more than two proteins are PEG-linked, separate the dumbbells via chromatographic methods known in the art. Where such separation is undesirable for any reason, delete or replace a free cysteine with another amino acid and proceed to step 4(b).
(6) For non-protein biologically-active R groups, exploit free sulfhydryl groups for attachment to the polymer X. Add free sulfhydryl groups to the molecule if necessary or desirable.
One skilled in the art might choose to modify, add or delete certain steps. For example, one might choose to react active proteins with a bifunctional-PEG and skip the monoPEGylation step.
Several dumbbell molecules of the present invention have been prepared. Published PCT Application No. WO 92/16221, which is incorporated herein by reference, sets forth the preparation of the following dumbbells prepared using bis-maleimido-PEG: 30 kDa TNF inhibitor homodumbbells, Il-2 inhibitor heterodumbbell, heterodumbbells which inhibit the classical pathway of the complement system, and IL-1ra and PDGF heterodumbbells.
Pharmaceutical compositions containing many of the conjugates or compounds (collectively, the xe2x80x9cconjugatesxe2x80x9d) of the present invention can be prepared. These conjugates can be in a pharmaceutically-acceptable carrier to form the pharmaceutical compositions of the present invention. The term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d as used herein means a non-toxic, generally inert vehicle for the active ingredient, which does not adversely affect the ingredient or the patient to whom the composition is administered. Suitable vehicles or carriers can be found in standard pharmaceutical texts, for example, in Remington""s Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980), incorporated herein by reference. Such carriers include, for example, aqueous solutions such as bicarbonate buffers, phosphate buffers, Ringer""s solution and physiological saline. In addition, the carrier can contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
The pharmaceutical compositions can be prepared by methods known in the art, including, by way of an example, the simple mixing of reagents. Those skilled in the art will know that the choice of the pharmaceutical carrier and the appropriate preparation of the composition depend on the intended use and mode of administration.
In one embodiment, it is envisioned that the carrier and the conjugate constitute a physiologically-compatible, slow-release formulation. The primary solvent in such a carrier can be either aqueous or non-aqueous in nature. In addition, the carrier can contain other pharmacologically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier can contain still other pharmacologically-acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the conjugate. Such excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dose or multi-dose form.
Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations can be stored either in a ready to use form or requiring reconstitution immediately prior to administration. The preferred storage of such formulations is at temperatures at least as low as 4xc2x0 C. and preferably at xe2x88x9270xc2x0 C. It is also preferred that such formulations containing the conjugates are stored and administered at or near physiological pH. It is presently believed that administration in a formulation at a high pH (i.e. greater than 8) or at a low pH (i.e. less than 5) is undesirable.
The manner of administering the formulations containing the conjugates for systemic delivery can be via subcutaneous, intramuscular, intravenous, oral, intranasal, or vaginal or rectal suppository. Preferably the manner of administration of the formulations containing the conjugates for local delivery is via intraarticular, intratracheal, or instillation or inhalations to the respiratory tract. In addition it may be desirable to administer the conjugates to specified portions of the alimentary canal either by oral administration of the conjugates in an appropriate formulation or device.
In another suitable mode for the treatment of osteoporosis and other bone loss diseases, for example, an initial intravenous bolus injection of TN inhibitor conjugate and IL-1 inhibitor conjugate is administered followed by a continuous intravenous infusion of TNF inhibitor conjugate and IL-1 inhibitor conjugate. For oral administration, the conjugate is encapsulated. The encapsulated conjugate can be formulated with or without pharmaceutically-acceptable carriers customarily used in the compounding of solid dosage forms. Preferably, the capsule is designed so that the active portion of the formulation is released at that point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional excipients can be included to facilitate absorption of the conjugate. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
Regardless of the manner of administration, the specific dose is calculated according to the approximate body weight of the patient. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, route of administration and the age, sex and medical condition of the pateint. In certiain embodiments, the dosage and administration is designed to create a preselected concentration range of the conjugate in the patient""s blood stream. For example, it is believed that the maintenance of circulating concentrations of TNF inhibitor and IL-1 inhibitor of less than 0.01 ng per mL of plasma may not be an effective composition, while the prolonged maintenance of circulating levels in excess of 10 xcexcg per mL may have undesirable side effects. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them without undue experimentation, especially in light of the dosage information and assays disclosed herein. These dosages may be ascertained through use of the established assays for determining dosages utilized in conjunction with appropriate dose-response data.
It should be noted that the conjugate formulations described herein may be used for veterinary as well as human applications and that the term xe2x80x9cpatientxe2x80x9d should not be construed in a limiting manner. In the case of veterinary applications, the dosage ranges should be the same as specified above.